A better understanding of how aging promotes malignant transformation may identify novel biomarkers for cancer detection and potential targets for future therapies. Preliminary studies have found that IRF8 expression decreases with increasing age in hematopoietic progenitor/stem cells (HPCs/HSCs) and in myeloid malignancies. In addition, previous murine studies suggest that silencing IRF8 promotes the development of "pre-leukemic" clones that transform into an acute myeloid leukemia (AML) with secondary genetic "hits."3,20,21 Therefore, decreased IRF8 expression in aging HPCs/HSCs may be an early pre-transforming event that leads to myeloid malignancies upon the acquisition of secondary "hits." Specific Aim 1. Determine if silencing IRF8 expression in human CD34+ cells promotes transformation into AML. In murine models, silencing IRF8 blocks monocytic differentiation, inhibits apoptosis, and leads to increased numbers of progenitor cells.4,18,19 Given the differences between the murine and human hematopoietic cells,22-25 we will silence IRF8 expression in human CD34+ cells and determine the in vitro and in vivo functional effects of this silencing. We hypothesize that silencing IRF8 expression in human CD34+ cells will block monocytic differentiation, inhibit apoptosis, and increase the number of progenitor cells. Furthermore, we hypothesize that NOD/SCID mice transplanted with human IRF8-deficient CD34+ cells will develop a MPD, which will develop into AML over time. Specific Aim 2. Investigate the prognostic significance of IRF8 expression in AML. Our preliminary analyses suggest that decreased IRF8 expression promotes in vitro chemoresistance and is associated with increasing age, unfavorable cytogenetics, and refractory disease in AML patients. Thus, we will examine IRF8 expression in flow-sorted leukemic blasts from AML patients with normal cytogenetics and determine whether IRF8 expression is associated with demographics, laboratory values, other prognostic factors, and clinical outcomes. We hypothesize that decreased IRF8 expression will be an independent poor prognostic factor for AML patients with normal cytogenetics. Long-term objectives. These experiments will advance our understanding of the functional and clinical significance of diminished IRF8 expression in normal and malignant hematopoietic cells. If decreased IRF8 expression is an early initiation event that promotes development of AML and other malignancies, IRF8-based assays will be developed to identify and examine these dysfunctional HPCs/HSCs and analyze associations between decreased IRF8 expression and risk of disease development. Furthermore, if decreased IRF8 expression is a poor prognostic factor, IRF8 expression will be incorporated into future studies as a prognostic factor, and we will develop studies to examine the underlying cause of driving the chemoresistance and potential therapeutic options to overcome it. PUBLIC HEALTH RELEVANCE: Preliminary studies have found that IRF8 expression decreases with increasing age in hematopoietic cells and may be associated with the development of leukemia. The proposed studies will examine the potential role of IRF8 as a biomarker of aging, malignant transformation, and AML prognosis. The results from these studies may lead to the development of novel IRF8-based assays that can be utilized to risk-stratify subjects prior to and after disease development, so that targeted preventative and therapeutic interventions can be initiated.